DESCRIPTION: Recombinant proteins are promising as therapeutics because they have specific functional properties that often cannot be adequately mimicked by small molecule drugs. However, the inability of many therapeutic proteins to concentrate at a disease site typically necessitates administration at relatively high doses, which often results in off-site effects and toxicity. Numerous protein engineering and controlled-release strategies have been investigated to enhance site-specific protein delivery, however, each is hindered by different mechanisms that can diminish protein activity. The primary goal of the proposed research is to create a facile approach to endow therapeutic proteins with targeting properties, without requiring a drug delivery vehicle or extensive protein engineering. Throughout nature, self-assembly of different functional proteins gives rise to supramolecular entities that can perform complex tasks via the co-integrated activity of each protein subunit. The long-term goal of our research program is to create multifunctional nanomedicines with precisely defined composition of therapeutic, targeting or diagnostic properties via self-assembly of different engineered proteins. To achieve this goal, the proposed research will create a trio of engineered peptide tags that selectively co-assemble into a heterogeneous a-helical coiled-coil via electrostatic and hydrophobic complementarity. Multifunctional nanomaterials will then be created through a three-step process: 1) genes encoding each of these peptide tags will be linked to genes encoding different functional protein ligands, 2) these recombinant fusion genes will be expressed by microbial hosts, and 3) the recovered fusion proteins will co-assemble into a precisely defined tripartite nanomaterial having function related to each co-integrated ligand upon mixing. Importantly, because the recombinant fusion tags mediating assembly are independent from the protein ligand itself, we envision that the functional protein component can be easily interchanged via DNA recombination, thus allowing for functional properties of the resulting nanomaterial to be precisely varied without altering its assembly. In Specific Aim 1, we will develop fusion proteins that assemble into a trifunctional nanomaterial via engineered peptide domains that selectively assemble into a heterogeneous trimeric a-helical coiled-coil. We will characterize influence of co-assembly on the bioactivity of each integrated ligand, as well as the serum stability of these assemblies. In Specific Aim 2, we will then assess the immunogenicity of peptide and fusion protein assemblies based on heterogeneous a-helical coiled-coils, expecting that they will be minimally immunogenic in the absence of an immunostimulatory adjuvant. Success of the proposed research will add significantly to the growing body of work utilizing a-helical coiled-coils to create multimeric protein nanoassemblies, by providing a general approach for modular nanomaterial design. In turn, these materials may provide the basis for nanomedicines with modular composition of therapeutic, targeting and diagnostic that can be precisely tailored to address a broad spectrum of diseases.